The present invention relates generally to isolated and purified proteins and nucleic acids which modulate TGF-xcex2 biological activity, including TGF-xcex2 signal transduction. More particularly, the present invention relates to an isolated and purified serine-threonine kinase receptors associated protein and an isolated and purified polynucleic acid encoding the same.
The publications and other materials used herein to illuminate the background of the invention, and in particular cases, to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the following text, and respectively grouped in the appended list of references.
Transforming growth factor xcex2""s (TGFxcex2) are a family of multifunctional cell regulatory factors produced in various forms by many types of cells (for review see Spom et al., J. Cell Biol. 105:1039, (1987)). Five different TGFxcex2""s are known, and the functions of two, TGFxcex2-1 and TGFxcex2-2, have been characterized in detail. TGFxcex2""s are the subject of U.S. Pat. Nos. 4,863,899; 4,816,561; and 4,742,003 which are incorporated by reference. TGFxcex2-1 and TGFxcex2-2 are publicly available through many commercial sources (e.g. R and D Systems, Inc. of Minneapolis, Minn.). These two proteins have similar functions and will be here collectively referred to as TGF-xcex2.
TGF-xcex2 binds to cell surface receptors possessed by essentially all types of cells, causing profound changes in them. In some cells, TGF-xcex2 promotes cell proliferation and in others it suppresses proliferation. A marked effect of TGF-xcex2 is that it promotes the production of extracellular matrix proteins and their receptors by cells (for review see Keski-Oja et al., J. Cell Biochem. 33:95 (1987); Massague, Cell 49:437 (1987); Roberts and Sporn in xe2x80x9cPeptides Growth Factors and Their Receptorsxe2x80x9d (Springer-Verlag, Heidelberg) (1989)).
While TGF-xcex2, has many essential cell regulatory functions, improper TGF-xcex2 activity can be detrimental to an organism. Since the growth of mesenchyme and proliferation of mesenchymal cells is stimulated by TGF-xcex2, some tumor cells may use TGF-xcex2 as an autocrine growth factor. Therefore, if the growth factor activity of TGF-xcex2 could be prevented, tumor growth could be controlled. In other cases the inhibition of cell proliferation by TGF-xcex2 may be detrimental, in that it may prevent healing of injured tissues. The stimulation of extracellular matrix production by TGF-xcex2 is important in situations such as wound healing. However, in some cases the body takes this response too far and an excessive accumulation of extracellular matrix ensues. An example of excessive accumulation of extracellular matrix is glomerulonephritis, a disease with a detrimental involvement of TGF-xcex2.
Pleiotropic responses to TGF-xcex2 are mediated via ligand-induced heteromeric complex formation by type I and type II receptors. Upon ligand binding, the type if receptor (Txcex2R-II), which is a constitutively active kinase, transphosphorylates the type I receptor (Txcex2R-I) and activates this kinase to propagate the signals to downstream effectors, termed SMAD proteins. See Massague et al., Trends Cell Biol. 7:187-192 (1997): Heldin et al., Nature 390:465-471 (1997). SMAD proteins can be classified according to their role in signaling by TGF-xcex2 family members. Pathway-restricted SMADs interact transiently with, and are phosphorylated by specific activated type I receptors. Smad2 and Smad3 mediate signaling by TGF-xcex2 and activin, whereas Smad1 and Smad5 are involved in BMP signaling. Smad4 is a common mediator of TGF-xcex2, activin and BMP signals. Recently Smad6 and Smad7 have been shown to function as inhibitors of these signaling pathways by interfering with the activation of pathway-restricted SMADs. Although the nature and mechanism of activation of TGF-xcex2 receptors at the cell surface has been described and the ester of potential regulators of TGF-xcex2 signaling continues to expand, little is known at the molecular level about the signaling mechanisms immediately downstream of the TGF-xcex2 receptors.
This lack of knowledge represents a serious deficiency in the art in view of the effects of cell regulatory factors such as TGF-xcex2 as described above. Therefore, further characterization of TGF-xcex2 signaling in vertebrates, particularly in mammals, and more particularly in humans is needed. A novel isolated and purified polypeptide having a role in the modulation of TGF-xcex2 signaling would have broad utility due to the various and multiple physiological and pathophysiological roles of TGF-xcex2, as described above.
The present invention contemplates an isolated and purified vertebrate serine-threonine kinase receptors associated protein (STRAP) which plays a role in the modulation of TGF-xcex2 biological activity. More preferably, a polypeptide of the invention is a recombinant polypeptide. Even more preferably, a polypeptide of the present invention comprises a mammalian STRAP. Even more preferably, a polypeptide, of the present invention comprises a human STRAP. Even more preferably, a polypeptide of the present invention comprises the amino acid residue sequence of SEQ ID NO:2.
The present invention also provides an isolated and purified polynucleotide that encodes a polypeptide that plays a role in the modulation of TGF-xcex2 biological activity. In a preferred embodiment, a polynucleotide of the present invention comprises a DNA molecule from a vertebrate species. A preferred vertebrate is a mammal. A preferred mammal is a human. More preferably, a polynucleotide of the present invention encodes a polypeptide designated STRAP. Even more preferred, a polynucleotide of the present invention encodes a polypeptide comprising the amino acid residue sequence of SEQ ID NO:2. Most preferably, an isolated and purified polynucleotide of the invention comprises the nucleotide base sequence of SEQ ID NO:1.
In another embodiment, the present invention provides an antibody immunoreactive with a STRAP polypeptide as described above. SEQ ID NO:1 and SEQ ID NO:2 set forth representative vertebrate nucleotide and amino acid sequences. Also contemplated by the present invention are antibodies immunoreactive with homologues or biologically equivalent STRAP polynucleotide and potypeptides found in other vertebrates. Preferably, an antibody of the invention is a monoclonal antibody. More preferably, the STRAP polypeptide comprises human STRAP. Even more preferably, the STRAP polypeptide comprises the amino acid residue sequence of SEQ ID NO:2.
In another aspect, the present invention contemplates a method of producing an antibody immunoreactive with STRAP as described above, the method comprising the steps of (a) transfecting a recombinant host cell with a polynucleotide that encodes a STRAP polypeptide having a TGF-xcex2 activity-modulating function; (b) culturing the host cell under conditions sufficient for expression of the polypeptide; (c) purifying the polypeptide; and (d) raising the antibody to the polypeptide. Preferably, the host cell is transfected with the polynucleotide of SEQ ID NO:1. Even more preferably, the present invention provides an antibody prepared according to the method described above. Also contemplated by the present invention is the use of homologues or biologically equivalent polynucleotides and polypeptides found in other vertebrates to produce antibodies.
Altermatively, the present invention provides a method of detecting a STRAP polypeptide as described above, wherein the method comprises immunoreacting the polypeptide with an antibody prepared according to the method described above to form an antibody-polypeptide conjugate, and detecting the conjugate.
In yet another embodiment, the present invention contemplates a method of detecting a messenger RNA transcript that encodes a STRAP polypeptide as described above, wherein the method comprises hybridizing the messenger RNA transcript with a polynucleotide sequence that encodes that polypeptide to form a duplex; and detecting the duplex. Altermatively, the present invention provides a method of detecting a DNA molecule that encodes a STRAP polypeptide as described above, wherein the method comprises hybridizing DNA molecules with a polynucleotide that encodes a STRAP polypeptide having a TGF-xcex2 activity-modulating function to form a duplex; and detecting the duplex.
In another aspect, the present invention contemplates an assay kit for detecting the presence of a STRAP polypeptide in a biological sample, where the kit comprises a first container containing a first antibody capable of immunoreacting with a vertebrate STRAP polypeptide having a TGF-xcex2 activity-modulating function, with the first antibody present in an amount sufficient to perform at least one assay. Preferably, an assay kit of the invention further comprises a second container containing a second antibody that immunoreacts with the first antibody. More preferably, the antibodies used in an assay kit of the present invention are monoclonal antibodies. Even more preferably, the first antibody is affixed to a solid support More preferably still, the first and second antibodies comprise an indicator, and, preferably, the indicator is a radioactive label or an enzyme.
In an alternative aspect, the present invention provides an assay kit for detecting the presence, in biological samples, of a STRAP polypeptide, the kits comprising a first container that contains a second polynucleotide identical or complementary to a segment of at least 10 contiguous nucleotide bases of a polynucleotide that encodes a STRAP polypeptide having a TGF-xcex2 activity-modulating function.
In another embodiment, the present invention contemplates an assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with a STRAP polypeptide, the kit comprising a first container containing a STRAP polypeptide having a TGF-xcex2 activity-modulating function that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay.
In still a further embodiment, this invention pertains to therapeutic methods based upon the TGF-xcex2 activity-modulating function of STRAP as described herein.
Thus, an aspect of the present invention pertains to the discovery of the novel STRAP protein and nucleic acid encoding the STRAP protein. Preferred nucleic acid and amino acid sequences for STRAP are described in SEQ ID NO:1 and SEQ ID NO:2.
It is another aspect of this invention that the novel STRAP protein acts in the TGF-xcex2 cascade to modulate TGF-xcex2 biological activity.
It is thus another aspect of this invention to provide a purified and isolated STRAP polypeptide having a TGF-xcex2 activity-modulating function.
The foregoing aspects and embodiments have broad utility given the biological significance of the TGF-xcex2 cascade, as is known in the art. By way of example, the foregoing aspects and embodiments are useful in the preparation of screening assays and assay kits that are used to identify compounds that affect or modulate TGF-xcex2 biological activity, or that are used to detect the presence of the proteins and nucleic acids of this invention in biological samples. Additionally, it is well known that isolated and purified polypeptides have utility as feed additives for livestock and further polynucleotides encoding the polypeptides are thus useful in producing the polypeptides.
Some of the aspects and objects of the invention having been stated hereinabove, other aspects and objects will become evident as the description proceeds, when taken in,connection with the accompanying Drawings and Examples as best described hereinbelow.